Truncated mammalian growth factor DNA sequence

ABSTRACT

A truncated mammalian growth factor, displaying homology to both basic and acidic fibroblast growth factor in a single polypeptide, is disclosed herein. The growth factor is substantially smaller (i.e. has fewer amino acid residues) than the full-length mammalian growth factor, has a higher affinity for fibroblast growth factor receptors than full-length K-FGF and basic fibroblast growth factor and increased mitogenic activity. Also disclosed herein are DNA sequences encoding the truncated growth factor, pharmaceutical formulations containing the truncated growth factor and methods to heal burns and wounds in a mammal by administering the pharmaceutical formulations.

The United States Government has rights to this invention by virtue ofgrant No. CA42568 from The National Cancer Institute.

This is a continuation of application Ser. No. 07/901,705, filed Jun.22, 1992, now abandoned, which in turn is a continuation-in-part of Ser.No. 07/806,771 filed Dec. 6, 1991, which in turn is a continuation ofSer. No. 07/177,506 filed Apr. 4, 1988, now abandoned, which in turn isa continuation of Ser. No. 07/062,925 filed Jun. 16, 1987, nowabandoned.

FIELD OF THE INVENTION

This invention pertains to a mammalian growth factor, pharmaceuticalformulations comprising said factor and methods for healing wounds orburns in mammals comprising administering said formulations.

BACKGROUND OF THE INVENTION

This invention pertains to a novel polypeptide having mammalian growthfactor activity and to methods for using it.

A variety of diffusible factors which stimulate the growth of cells in ahormone-like manner are generally called "growth factors". Growthfactors are often present in serum and have also been isolated from avariety of organs. They are protein molecules (or groups of suchmolecules) and in all known cases they interact with specific cellsurface receptors to promote cellular growth and/or differentiation.Growth factors vary in their tissue specificity, i.e. some interact onlywith specific cell types, while others are active on a wider cell typerange.

Among the best known groups of mammalian growth factors are: (1)platelet derived growth factor (PDGF), released from platelets; (2)epidermal growth factor (EGF); (3) hematopoietic growth factors(including interleukins 1, 2, and 3), required for growth anddifferentiation of lymphocytes, and colony stimulating factors (CSF),promoting growth and differentiation of hematopoietic stem cells; (4)angiogenic (literally "blood-vessel-forming") growth factors, such asthe fibroblast growth factors (FGF) believed to promote growth andorganization of endothelial cells into new blood vessels; (5)miscellaneous growth factors released by tumor cells.

Two well-characterized angiogenic factors are basic and acidicfibroblast growth factors (FGF), believed to be most important in Vivofor endothelial cell growth. However, neither basic FGF nor acidic FGFhas proven useful as pharmaceutical agents for promotion of woundhealing. Several factors may contribute to the unsuitability of basicFGF and acidic FGF as pharmaceutical agents. Neither factor issufficiently stable for effective pharmaceutical formulation. Basic FGFdemonstrates restricted interaction with FGF receptors in vitro, andthus cannot be expected to interact with all FGF receptors in vivo.Finally, basic FGF and acidic FGF have thus far proven ineffective inanimal models.

Co-pending U.S. patent application Ser. No. 07/806,791 filed Dec. 6,1991 discloses an angiogenic mammalian growth factor isolated fromKaposi's Sarcoma cells and having substantial homology to each of acidicand basic fibroblast growth factor in a single polypeptide. The growthfactor protein comprises 176 amino acid residues and is a mature(secreted) glycoprotein. This growth factor has variously been calledK-FGF or FGF-4, and it has shown promising results as a wound healingagent in preclinical studies in an ischemic rabbit ear model. In such amodel, K-FGF promoted wound healing better than basic or acidic FGF.

Growth factors are believed to promote wound healing. For example, EGFpresent in saliva is believed to accelerate wound healing in mice.Schultz G.S et al. (Science 232:350-352, 1986) report that transforminggrowth factor (TGF)-alpha and vaccinia virus growth factor (VGF), bothof which are substantially homologous to EGF, accelerated epidermalwound healing in pigs when topically applied to second degree burns andwere significantly more active than EGF.

Of the above-mentioned growth factors, the angiogenic growth factorswould be particularly useful as wound healing agents because of theirability to promote the formation and growth of new blood vessels.

OBJECTS OF THE INVENTION

It is an object of the present invention to provide a novel growthfactor useful as a wound healing agent in mammals.

Another object of the present invention is to provide a mammalian growthfactor with increased biologic activities.

Yet another object of the present invention is to provide novelpharmaceutical formulations and methods for promoting wound healing inmammals.

A still further object of the present invention is to provide atruncated mammalian growth factor protein having substantial homology toeach of acidic and basic fibroblast growth factor protein in a singlepolypeptide and having substantially higher specific activity than K-FGFprotein.

SUMMARY OF THE INVENTION

The present invention pertains to a previously unknown form of truncatedmammalian growth factor protein having substantial homology to each ofbasic and acidic fibroblast growth factor proteins in a singlepolypeptide chain, said truncated mammalian growth factor beingsubstantially smaller than the full-length mammalian growth factor (thetruncated protein is hereinafter referred to as truncated K-FGF orK-FGF-140).

In another aspect, the present invention provides a polypeptide havingthe amino acid sequence (SEQ. ID. NO. 1): ##STR1##

In yet another aspect, the present invention provides a pharmaceuticalformulation for treating a mammal suffering from wounds or burnscomprising truncated K-FGF and a pharmaceutically acceptable carrier ordiluent.

A still further aspect of the present invention involves a method forhealing wounds or burns in a mammal in need of such treatment byadministration of an effective amount for wound or burn healing oftruncated K-FGF.

A still further aspect of the present invention provides an isolated DNAhaving the sequence (SEQ. ID. NO. 2):

    __________________________________________________________________________    GCG GCC GTC CAG AGC GGC GCC GGC GAC TAC CTG CTG GGC  39                       ATC AAG CGG CTG CGG CGG CTC TAC TGC AAC GTG GGC ATC  78                       GGC TTC CAC CTC CAG GCG CTC CCC GAC GGC CGC ATC GGC 117                       GGC GCG CAC GCG GAC ACC CGC GAC AGC CTG CTG GAG CTC 156                       TCG CCC GTG GAG CGG GGC GTG GTG AGC ATC TTC GGC GTG 195                       GCC AGC CGG TTC TTC GTG GCC ATG AGC AGC AAG GGC AAG 234                       CTC TAT GGC TCG CCC TTC TTC ACC GAT GAG TGC ACG TTC 273                       AAG GAG ATT CTC CTT CCC AAC AAC TAC AAC GCC TAC GAG 312                       TCC TAC AAG TAC CCC GGC ATG TTC ATC GCC CTG AGC AAG 351                       AAT GGG AAG ACC AAG AAG GGG AAC CGA GTG TCG CCC ACC 390                       ATG AAG GTC ACC CAC TTC CTC CCC AGG CTG TGA         423                       __________________________________________________________________________

A still further aspect of the present invention provides a truncatedK-FGF protein characterized by (i) a molecular weight of about 14,000Daltons; and (ii) an average FGF-receptor binding affinity of about9.5×10⁻¹¹ M.

These and other aspects of the present invention will be apparent tothose of ordinary skill in the art in light of the present description,claims and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram depicting the amino acid sequence of the full-lengthK-FGF protein and the amino acid sequence of the truncated protein ofthe present invention, K-FGF-140.

FIG. 2 is an autoadiograph of a sodium dodecyl sulfate polyacrylamidegel electrophoretic (SDS-PAGE) analysis of immunoprecipitated K-FGFforms produced in COS cells transfected with either wild type K-FGF orK-FGF-140 DNA or with a mutated K-FGF cDNA which expresses anunglycosylated form of K-FGF that is processed to produce K-FGF-140.

FIG. 3 is an autoradiograph of an SDS-PAGE analysis of the elution ofK-FGF and K-FGF-140 from heparin affinity columns.

FIG. 4 is a graph showing the stimulation of DNA synthesis in quiescentBALB/c-3T3 cells by recombinant K-FGF and K-FGF-140.

FIGS. 5 (A and B) are graphs depicting a competition assay of theability of K-FGF and K-FGF-140 to displace labeled basic fibroblastgrowth factor (bFGF) binding to Chinese Hamster Ovary (CHO) cellsexpressing the FGF receptor 1 (flg) or 2 (bek).

FIG. 6 (A-D) are a series of graphs depicting competition assays betweenK-FGF and K-FGF-140 for receptors on CHO cells expressing the FGFreceptor 1 (flg) or 2 (bek).

FIG. 7 is a graph depicting a Scatchard analysis of K-FGF and K-FGF-140binding to CHO cells expressing the FGF receptor 1 (bek).

DETAILED DESCRIPTION OF THE INVENTION

All patent applications, patents and literature references mentioned inthe specification are incorporated by reference in their entirety. Inthe case of inconsistencies, the present disclosure, includingdefinitions, shall prevail.

The present inventors have surprisingly found a truncated form of theK-FGF protein which demonstrates substantially increased activity overthat of either full-length K-FGF, basic FGF, or acidic FGF. Thetruncated K-FGF protein is not glycosylated and is substantially smaller(i.e. has fewer amino acid residues) than the full-length K-FGF protein.The truncated protein has a higher affinity for fibroblast growth factorreceptors than either the mature, full-length K-FGF protein or bFGF. Inaddition, the truncated protein has a higher affinity for heparin thanfull-length K-FGF and increased mitogenic (i.e. growth promoting)activity. It is expected that, due to these increased biologicalactivities, the truncated protein of the present invention will alsohave increased wound healing activity.

"Substantially smaller" refers to the fact that the truncated K-FGFprotein of the invention contains about 140 amino acid residues ascontrasted with the 176 amino acid residues that are present in thefull-length mature, secreted K-FGF protein.

"K-FGF-140" is defined herein as the unglycosylated, truncated mammaliangrowth factor protein of the present invention.

"K-FGF" is defined herein as the full-length mature human growth factorhaving a molecular weight of about 18,000 Daltons (non-glycosylated)comprising 176 amino acid residues as disclosed in U.S. patentapplication Ser. No. 07/806,791 filed Dec. 6, 1991.

"Mitogenic activity" in reference to the biological activity of thetruncated protein of the present invention is defined herein as theability of the protein to induce DNA synthesis and proliferation ofcells in culture.

"Substantial homology to each of acidic and basic fibroblast growthfactors" is defined herein as having regions of identity (either exactor by conservative substitution) to said growth factors as shown inTable 1 below.

K-FGF-140 was discovered during studies on the effect of glycosylationon the secretion of full-length K-FGF. Simian COS cells that weretransfected with a plasmid encoding the full-length human K-FGF proteinand incubated with tunicamycin (an inhibitor of N-linked glycosylation),accumulated an unglycosylated K-FGF protein within the cells ofapproximately 18,000 Daltons (the expected size of the unglycosylatedfull-length K-FGF protein). Surprisingly, only proteins of 12,000-14,000Daltons were detected in the culture medium (i.e. were secreted). Thiswas more clearly shown using a K-FGF cDNA mutated in such a way toexpress a protein in which amino acid 38 (Threonine) of the full lengthK-FGF precursor protein was replaced by Alanine. This protein cannot beglycosylated. COS cells transfected with a plasmid encoding this mutatedform of K-FGF also accumulate within the cells an unglycosylated K-FGFprotein of approximately 18,000 daltons, but produce in the medium onlyforms of 12,000-14,000 daltons. Apparently the removal of the sugarresidues exposes sites on the K-FGF molecule that are very susceptibleto cleavage by cellular proteases located on the cell surface. Thus theprotein is cleaved to produce these smaller forms as soon as it becomesexternalized. It has been determined that the 14,000 Dalton species isK-FGF-140, a truncated form of the full-length K-FGF protein.

K-FGF-140 retains the same regions of homology to acidic and basic FGFas the full-length K-FGF protein (as shown in Table 1 below) but hasincreased biological activity.

                                      TABLE 1                                     __________________________________________________________________________    BOVINE BASIC FIBROBLAST GROWTH FACTOR                                         67'AAQPKEAAVQSGAGDYLLG--IKRLRRLYCNVGIGFHLQALPDGRIGGAHADTRDSL--LEL              ##STR2##                                                                     119'SPVERGVVSIFGVASRFFVAMSSKGKLYGSPFFTDECRFKEILLPNNYNAYESYKYPGMF               ##STR3##                                                                     179'IALSKNGKTKKGNRVSPTMKVTHFLPRL                                               ##STR4##                                                                     BOVINE ACIDIC FIBROBLAST GROWTH FACTOR                                        67'AAQPKEAAVQSGAGDYLLGIKRLRRLYCNVGIGFHLQALPDGRIGGAHADTRDSL--LELS               ##STR5##                                                                     120'PVERGVVSIFGVASRFFVAMSSKGKLYGSPFFTDECTFKEILLPNNYNAYESYKYPGM----             ##STR6##                                                                     178'FIALSKNGKTKKGNRVSPTMKVTHFLPRL                                              ##STR7##                                                                     __________________________________________________________________________     A = Ala; R = Arg; N = Asn; D = Asp; C = Cys; Q = Gln; E = Glu; G = Gly; H     = His; I = Ile; L = Leu; K = Lys; M = Met; F = Phe; P = Pro; S = Ser; T =     Thr; W = Trp; Y = Tyr; V = Val.                                          

In Table 1, the N-terminal amino acid (alanine) of K-FGF-140 is residue67 of the full-length K-FGF protein. Two dots between a particular setof amino acid residues indicate exact identity between the truncatedgrowth factor of the present invention and either one of basic (SEQ. ID.NO. 3) and acidic FGF (SEQ. ID. NO. 4), and one dot indicates that therehas been a conservative substitution, e.g. substitution of the same typeof amino acid such as phenyl-alanine substituted for tyrosine. Inaddition, the amino acid sequence of the truncated growth factor of thepresent invention are number 67'-206', while the FGF sequences arepresented as 1"-146" and 1"-141" for basic and acidic FGF, respectively.That is to say residues 1"-146" comprise the sequence of basic FGF,while residues 1"-141" comprise acidic FGF.

As shown in Example 5 below, the truncated protein had mitogenicactivity that is 4-5 times greater (i.e. increased DNA synthesis andcell proliferation activity) than the full-length K-FGF protein as shownby its ability to induce proliferation of 3T3 cells at concentrations4-5 times lower than those of K-FGF. The truncated K-FGF protein alsohas a higher affinity for two of the FGF receptors than either thefull-length K-FGF protein or basic fibroblast growth factor (bFGF).

The growth factor of the present invention can be obtained from themedium of cells transfected or transformed by the "wild type" orfull-length K-FGF gene that have been cultivated in the presence ofglycosylation inhibitors, such as tunicamycin. Alternatively, K-FGF-140can be obtained from the medium of cells transfected or transformed by amutated K-FGF cDNA that produces a protein incapable of beingglycosylated, as the one described above, or preferably by using asuitable DNA construct to transform or transfect a eukaryotic, plant, orbacterial cell (e.g. E. coli), the latter described in Example 2 below.The wild type full-length gene can be obtained as described inco-pending U.S. patent application Ser. No. 7/806,791 filed Dec. 6,1991. Alternatively the DNA sequence can be used to chemicallysynthesize the K-FGF-140 gene using techniques well known in the art.

The DNA encoding the growth factor of the present invention can becloned and the protein can be expressed in any eukaryotic or prokaryoticsystem known in the art. Non-limiting examples of suitable eukaryoticexpression systems include yeast expression vectors (described by Brake,A. et al., Proc. Nat. Acad. Sci. USA 81: 4642-4646, 1984), Polyoma virusbased expression vectors (described in Kern, F. G. et al. Gene 43:237-245, 1986) Simian virus 40 (SV40)-based expression vectors in COS-1Simian cells (as described in Gerbing, M.J. et al. Nature 293: 620-625,1981) and baculovirus (insect)-based expression vectors (described inU.S. Pat. No. 4,745,051, issued May 17, 1988 and U.S. Pat. No.4,879,232, issued Nov. 7, 1989). An example of a procaryotic expressionsystem (e.g. E. coli) is presented below in Example 2 and an example ofa eukaryotic expression system (e.g. COS cells) is presented below inExample 3. Particularly preferred expression vectors include E. coli,simian COS cells and baculovirus (insect) cells.

The DNA encoding the truncated mammalian growth factor of the presentinvention may be modified without changing the primary sequence of theencoded polypeptide in order to increase the efficiency of itsproduction. One such example is presented in Example 2 below where ATnucleotides were incorporated into the 5' end of the molecule forcloning into E. coli. In addition, an ATG encoding methionine, was alsoadded to the 5' end of the DNA. Other modifications for cloning andexpression in other systems are known in the art and are within thescope of the present invention.

The DNA sequence (SEQ. ID. NO. 5) of K-FGF-140 is as follows: ##STR8##

The polypeptide of the present invention can be purified by any one ofthe many techniques that are well known in the art for use inconjunction with the expression system to produce the polypeptide. Forexample, when expressing the protein in E. coli, a purificationprocedure such as that disclosed in Example 2 below may be used.

The truncated K-FGF mammalian growth factor of the present invention canbe employed as a wound-healing agent for various mammalian wounds, suchas decubitus ulcers or burns. When employed as a wound or burn healingagent, the growth factor of the present invention may be administered toa mammal in need of such treatment orally, parenterally, or preferably,topically, directly to the affected area in amounts broadly rangingbetween about 10 nanograms and about 10 micrograms per dose. The numberof treatments required to treat a particular wound or burn and theduration of treatment can vary from individual to individual dependingupon the severity of the wound or burn. A typical treatment wouldcomprise 1 or 2 topical applications per day, that are applied directlyto the surface of the wound or burn.

The growth factor of the present invention can be prepared inpharmaceutical formulations or dosage forms to be used as a wound orburn healing agent. Pharmaceutical formulations containing the mammaliangrowth factor of the present invention (or physiologically acceptablesalts thereof) as at least one of the active ingredients may alsocontain pharmaceutically-acceptable carriers, diluents, fillers, saltsand other materials well-known in the art depending upon the dosage formutilized. For example, parenteral dosage forms may comprise aphysiologic, sterile saline solution. Topical dosage forms may comprisefor example, lanolin, hydroxymethyl cellulose or propylene glycol. In analternative embodiment, the mammalian growth factor of the presentinvention may be mixed with antibiotic creams (such as Silvadene, MarionLaboratories, Kansas City, Mich., Achromycin, Lederle Laboratories,Pearl River, N.Y., or Terramycin, Pfipharmecs, New York, N.Y.)well-known in the art.

As will be understood by those of ordinary skill in the art, thepharmaceutical formulations or dosage forms of the present inventionneed not contain an effective amount of the truncated protein of thepresent invention as such effective amounts can be achieved byadministering a plurality of formulations or dosage forms.

Although the truncated K-FGF growth factor of the present invention isparticularly useful as a wound or burn healing agent it also can beemployed as an agent to promote the growth of cells in tissue cultureand/or as a partial serum substitute. The growth-promoting properties oftruncated K-FGF are illustrated in Example 5 below.

The invention is described further below in specific working exampleswhich are intended to illustrate the present invention without limitingits scope.

EXAMPLE 1

AMINO ACID SEQUENCE OF THE HUMAN K-FGF PRECURSOR PROTEIN

The amino acid sequence of K-FGF and K-FGF-140 are shown in FIG. 1. InFIG. 1, arrows under the sequence indicate the sites of cleavage of themature, secreted form of K-FGF. Asterisks indicate the glycosylationsignal. The result of the mutation introduced in the cDNA to eliminateglycosylation is indicated above the asterisks (Threonine to Alanine).The [sign indicates the site of cleavage which generates K-FGF-140.

EXAMPLE 2

CONSTRUCTION AND EXPRESSION VECTOR FOR K-FGF-140

The K-FGF-140 cDNA (which was mutated at the glycosylation site) wasexpressed in COS cells using media conditions that allowed tritiatedleucine to be incorporated into the expressed protein. Theleucine-labeled protein was purified by precipitation with a polyclonalantibody raised against full-length K-FGF. The amino terminus of thepurified K-FGF-140 protein was sequenced using a protein sequencer(Applied Biosystems model 470A). Tritium was found in several cycles andthese cycles were assigned as leucine residues. There was a majorsequence and a minor sequence. By a process of elimination the majorsequence was identified as starting at residue 67 of the K-FGFfull-length sequence (Delli-Bovi et al. Cell 50: 729-37 1987, Delli-Boviet al. 1988 Molecular and Cellular Biology 8: 2933-41). The sequence ofthis truncated protein is illustrated in FIG. 1.

A variety of different expression vectors may be used to produce theK-FGF-140 protein in E. coli. A bacterial expression vector was designedand constructed encoding the K-FGF-140 protein under the control of thebacteriophage lambda pL promoter and the cII ribosome binding site.

The full-length cDNA sequence of K-FGF was altered using site directedmutagenesis (T.A. Kunkel et al. (1987) Methods in Enzymol., Vol. 154,pages 367-382) to delete the sequence for the first 66 amino acids andplace an initiator methionine in front of residue 67. It was also founddesirable to change the codon usage pattern (using site directedmutagenesis) at the start of the truncated sequence to codons containingmore Adenine or Thymidine. The sequence changes that were made areillustrated in Table 2 below.

                                      TABLE 2                                     __________________________________________________________________________    Original SequenceGCGGCCGTCCAGAGCGGCGCCGGCGAC. . . SEQ ID NO:6:                 ##STR9##                                                                     Amino AcidMetAlaAlaValGlnSerGlyAlaGlyAsp. . . SEQ ID NO:8:                    __________________________________________________________________________

In Table 2, the nucleotides which were changed are underlined. None ofthe changes resulted in a change in the amino acids sequence of theprotein.

Other changes to more favorable codons or changes further into thesequence could also have been made. This AT rich sequence at the startof the gene was found to optimize the amount of K-FGF-140 proteinexpressed in E. coli B4.

The K-FGF-140 gene was expressed in E. coli. Expression of the gene wasaccomplished by growing cells at 30° C. (the permissive temperature forthe temperature sensitive lambda repressor). The culture was thenshifted to 40° C. where the lambda repressor fails to repress the pLlambda promoter and maintained at this temperature for 3 hours. Cellswere harvested by centrifugation and stored at -80° C. The bacterialcells were broken in the presence of break buffer (6.0 g Tris adjustedto pH 7.0 with HCl, 1.9 g EDTA, 1.7 g PMSF, 1.0 g pABA all in 1 L water)in a homogenizer (Gaulin model 15). The cells were passed three timesthrough the homogenizer at a pressure differential of 8000-9000 poundsper square inch (PSI). The broken cell paste was frozen in liquidnitrogen and stored at -80° C.

Most of the K-FGF-140 protein was found in the insoluble fraction in thecell lysate and was harvested by centrifugation. The growth factor wasextracted from the centrifugation pellet by suspension in extractionbuffer (50 mM Tris pH 7.5, 200 mM MgCl₂). The extract was centrifugedand K-FGF-140 was found in the soluble fraction. This fraction wasloaded onto a heparin Toyopearl (Tosohaas) column, and the bufferexchanged with 0.5M NaCl 50 mM Tris pH 7.5 followed by 0.5M NaCl, 20 mMNa phosphate pH 7.5. Finally, the K-FGF-140 protein was eluted with agradient of 0.5-1.75 NaCl in 20 mM Na phosphate pH 7.5. The protein wasfound to elute at a salt concentration of about 1.55 M NaCl whereasfull-length K-FGF elutes at about 1.15M NaCl.

EXAMPLE 3

IMMUNOPRECIPITATION ANALYSIS OF THE K-FGF FORMS PRODUCED IN COS CELLSTRANSFECTED WITH THE GLYC-K-FGF-140 cDNA

COS cells were transfected with the 91203B expression plasmid (describedin Delli-Bovi et al. (1987) Cell, Vol. 50, pages 729-737) containingeither the full-length human K-FGF cDNA or a mutated cDNA encoding aprotein lacking the N-linked glycosylation signal (glyc(-)cDNA). 40hours later the cells were labelled with ³⁵ S-methionine for 8 hours, inthe presence (+) or absence (-) of tunicamycin, a drug that inhibitsN-linked glycosylation. Labelled proteins from either the cell lysate(indicated as L) or medium (indicated as M) were immunoprecipitated withanti-K-FGF rabbit antibodies and electrophoresed on SDS-PAGE. The gelwas then subjected to autoradiography. The results are shown in FIG. 2.

In FIG. 2, M.W. markers are indicated on the right. It can be seen thatthe cell transfected with the glyc(-)cDNA expressed in the cell lysate aprotein of apparent M.W. of 18,000 Daltons, identical to the oneproduced by the wild-type K-FGF DNA in the presence of tunicamycin. Thisprotein cannot however, be detected in the culture medium, where onlytwo bands of MW 12,000-14,000 were seen.

EXAMPLE 4

ELUTION OF K-FGF-140 FROM HEPARIN AFFINITY COLUMNS

Conditioned Medium labeled with ³⁵ S-methionine produced from COS cellstransfected with either of K-FGF or glyc-cDNAs (i.e. K-FGF-140) wasabsorbed to Heparin-Sepharose columns and eluted with increasing saltconcentrations. Fractions were immunoprecipitated with anti-K-FGFantibodies, and electrophoresed on SDS-PAGE to identify the K-FGFproteins. The results are shown in FIG. 3.

It can be seen that all or most of K-FGF eluted at 1.1M NaCl while thetruncated K-FGF forms eluted with a peak at 1.3-1.6M NaCl.

EXAMPLE 5

STIMULATION OF DNA SYNTHESIS IN QUIESCENT BALB/c-3T3 CELLS BY HUMANRECOMBINANT K-FGF OR BY RECOMBINANT K-FGF-140

BALB/c-3T3 cells were incubated for two days in medium containing 0.5%serum, at which point cells were treated with different concentrationsof K-FGF or K-FGF-140. 18 hours later the cells were labeled with ³H-thymidine (1 μCi/ml) for 6 hours. Radioactivity incorporated intocellular DNA was counted after trichloroacetic acid (TCA) precipitation.The results are shown in FIG. 4.

In FIG. 4, 0.5%=negative control, 10%=cells stimulated with 10% serum.As can be seen from the data in FIG. 5, 0.1 ng of K-FGF-140 was capableof producing the same stimulation of DNA synthesis as that of 0.5 ng ofK-FGF. Furthermore, maximum stimulation in ³ H-thymidine uptake occurredusing 1ng/ml of K-FGF-140 and 5ng/ml full-length K-FGF. Treatment with1ng/ml of K-FGF-140 led to a greater amount of cell proliferation thanall other additions, including 10% serum.

EXAMPLE 6

RECEPTOR BINDING

To study the affinity of K-FGF-140 for FGF receptors, the ability ofK-FGF and K-FGF-140 to compete with ¹²⁵ I-labeled basic fibroblastgrowth factor (bFGF) for binding to CHO 4-1 cells expressing FGFreceptor-1 (Mansukhani, A. et al. (1992) Proc. Natl, Acad. Sci. USA,Vol. 89, pages 3305-3309) (A) or to CHO 3-7.5 cells expressing the FGFreceptor-2 (Mansukhani, A. et al. (1990) Proc. Natl. Acad. Sci. USA,Vol. 87, pages 4378-4382) (B) was performed. Cells (1×10⁶ cells/35 mmdish) were incubated at 4° C. with Dulbecco's modified EAGLE's medium(DMEM) containing 0.15% gelatin, 25 mM Hepes (pH 7.4), Heparin (10μg/ml), ¹²⁵ I-labeled bFGF (4 ng/ml s.a. 3.2×10¹⁷ cpm/mole,Collaborative Research) and the indicated concentration of unlabeledbFGF, K-FGF or K-FGF-140. After 2 hours the cells were washed with 2MNaCl buffered at pH 7.4 to remove growth factor bound to the matrix, andwith 2M NaCl buffered at pH 4.0 to remove the ligand bound to highaffinity receptors. The amount of ¹²⁵ I-labeled bFGF bound to highaffinity receptors was determined. The results are shown in FIG. 5 (Aand B).

In the CHO clone expressing the FGF receptor 1 (FIG. 5A) the data showthat about 8 times more K-FGF than bFGF or K-FGF-140 was needed tocompete for the binding of ¹²⁵ I-bFGF; in the CHO clone expressing theFGF receptor 2 (FIG. 5B), K-FGF-140 was more efficient than K-FGF andbFGF in competing for the binding of ¹²⁵ I-labeled bFGF. In this casethe affinity of K-FGF-140 for the receptors was about three times higherthan that of bFGF or full-length K-FGF.

EXAMPLE 7

COMPETITION BETWEEN K-FGF AND K-FGF-140 FOR RECEPTOR BINDING

Clones CHO 4-1 expressing FGF receptor-1 (FIG. 6A and FIG. 6C) or CHO3-7.5 expressing FGF receptor-2 (FIG. 6B and FIG. 6D) were incubatedwith ¹²⁵ I-labeled K-FGF-140 (11 ng/ml, specific activity 7.7×10¹⁶cpm/mole) (FIG. 6A and B) or with ¹²⁵ I-labeled K-FGF (8 ng/ml, specificactivity 9.9×10¹⁶ cpm/mole) (FIG. 6C and D), Heparin (10 ug/ml) and theindicated concentration of unlabeled K-FGF or K-FGF-140. After 2 hoursat 4° C. the medium was removed, the cells were washed with ice coldTris and were lysed in 0.6% SDS/50mM.Tris/HCl pH 7.4, 0.15M NaCl, 5mM.EDTA, and the cell associated radioactivity was determined. The dataare expressed as % of inhibition of Iodine labeled growth-factor bindingby the indicated amount of unlabeled growth factor.

The data presented in FIG. 6 show that K-FGF-140 had a higher affinityfor both FGF (FIGS. 6A and 6C and FIGS. 6B and 6) receptors thanfull-length K-FGF protein.

EXAMPLE 8

SCATCHARD ANALYSIS OF K-FGF AND K-FGF-140 BINDING TO CHO CELLSEXPRESSING THE FLG RECEPTOR

Scatchard analysis of the binding of K-FGF and K-FGF-140 was performedon CHO 4-1 cells expressing the FGF receptor-1 as follows. Cells at4.8×10⁵ /35mm dish were incubated at 4° C. with DMEM containing 0.15%gelatin, 25 mM. Hepes(pH 7.4), Heparin (10 ug/ml) and variousconcentration of ¹²⁵ I-labeled K-FGF or K-FGF-140 from 0.15 to 20 ng/ml.After 2 hours the medium was removed, the cells were washed with icecold Tris and ¹²⁵ I-labeled K-FGF or K-FGF-140 bound to high affinityreceptors was removed by extraction in 0.6%SDS/50mMTris/HCl pH 7.4, 0.15mM NaCl, 5 mM EDTA. Non-specific binding was obtained using the sameamount of growth factor on parental CHO DG44 cells that do not expressFGF receptors. The results are shown in FIG. 7.

In FIG. 7, Scatchard analysis of binding to high affinity receptors gavea straight line, indicating a single class of binding sites. The dataalso indicate that the average binding affinity of K-FGF-140 for the flgreceptor is about 9.5×10⁻¹¹ M or about three times higher than that offull-length K-FGF which has an average binding affinity of about28.5×10⁻¹¹ M.

EXAMPLE 9

WOUND HEALING ASSAY

K-FGF-140 will be assayed in an ischemic wound healing system. For thispurpose the rabbit ear ischemic model of dermal ulcers, in which healingof these ulcers is retarded because of induced ischemia (reduced bloodflow) is used. After wounding (6 mm wounds) K-FGF-140 is applied eitherin an isotonic buffer or in a gel, applied in a single dose (1-5 μg),and compared to untreated controls, or wounds treated with K-FGF orbFGF. At various days after the beginning of the experiment (up to day7-10) the extent of wound healing is determined by measuring a) newepithelium formed at the gap of epithelia tissue at the beginning andend of the experiment) by histological cross sections; b) the gapbetween the two edges of the granulation tissues; and c) formation ofnew granulation tissue as measured by staining of immature vs. maturecollagen. These techniques are described in Ahn, S. T. and Mustoe, T. A.Annals Plastic Surgery 24: 17-23 (1990) and Mustoe, T. A., Pierce G. F.,Morishima, C. and Deuel, T.P. J. Clinical Invest. 87: 694-703 (1991).

From the in vitro experiments present above showing that K-FGF-140 hashigher potency and receptor affinity then K-FGF, it is expected thatK-FGF-140 will prove effective at accelerating wound healing in thesystem, and will prove more potent (effective at lower concentration,faster response) than K-FGF or bKFGF.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 8                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 140                                                               (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE:                                                           (A) DESCRIPTION: Protein                                                       (iii) HYPOTHETICAL: No                                                       (iv) ANTI-SENSE: No                                                           (ix) FEATURE:                                                                 (A) NAME/KEY:                                                                 (B) LOCATION:                                                                 (C) IDENTIFICATION METHOD:                                                    (D) OTHER INFORMATION: This sequence can be                                   found on page 4, lines 6-33, in the                                           application, as filed.                                                        (x) PUBLICATION INFORMATION:                                                  (A) AUTHORS:                                                                   (B) TITLE:                                                                   (C) JOURNAL:                                                                  (D) VOLUME:                                                                   (E) ISSUE:                                                                    (F) PAGES:                                                                    (G) DATE:                                                                     (H) DOCUMENT NUMBER:                                                          (I) FILING DATE:                                                              (J) PUBLICATION DATE:                                                         (K) RELEVANT RESIDUES IN SEQ ID NO: 1-140                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       AlaAlaVa lGlnSerGlyAlaGlyAspTyrLeuLeuGlyIleLysArg                             151015                                                                        LeuArgArgLeuTyrCysAsnValGlyIleGlyPheHisLeuGlnAla                              20 2530                                                                       LeuProAspGlyArgIleGlyGlyAlaHisAlaAspThrArgAspSer                              354045                                                                        LeuLeuGluLeuSerProValGluA rgGlyValValSerIlePheGly                             505560                                                                        ValAlaSerArgPhePheValAlaMetSerSerLysGlyLysLeuTyr                              657075 80                                                                     GlySerProPhePheThrAspGluCysThrPheLysGluIleLeuLeu                              859095                                                                        ProAsnAsnTyrAsnAlaTyrGluSerTyrLysTyrProGl yMetPhe                             100105110                                                                     IleAlaLeuSerLysAsnGlyLysThrLysLysGlyAsnArgValSer                              115120125                                                                     ProThr MetLysValThrHisPheLeuProArgLeu                                         130135140                                                                     (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 423                                                               (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii ) MOLECULE TYPE:                                                          (A) DESCRIPTION: Genomic DNA                                                  (iii) HYPOTHETICAL: No                                                        (iv) ANTI-SENSE: No                                                           (ix) FEATURE:                                                                 (A) NAME/KEY:                                                                 (B) LOCATION:                                                                 (C) IDENTIFICATION METHOD:                                                    (D) OTHER INFORMATION: This sequence can be                                   found on page 5, lines 3-13, in the                                           application, as filed.                                                        (x) PUBLICATION INFORMATION:                                                   (A) AUTHORS:                                                                 (B) TITLE:                                                                    (C) JOURNAL:                                                                  (D) VOLUME:                                                                   (E) ISSUE:                                                                    (F) PAGES:                                                                    (G) DATE:                                                                     (H) DOCUMENT NUMBER:                                                          (I) FILING DATE:                                                              (J) PUBLICATION DATE:                                                         (K) RELEVANT RESIDUES IN SEQ ID NO: 1-423                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                      GCGGCCGTCCAGAGCGGCGCCGGCGACTACCTGCTGGGC39                                     ATCAAGCGGCTGCGGCGGCTCTACTGCAACGTGGGCATC78                                     GGCTTCCACCTCCAGGCGCTCCCCGACGGCCGCATC GGC117                                   GGCGCGCACGCGGACACCCGCGACAGCCTGCTGGAGCTC156                                    TCGCCCGTGGAGCGGGGCGTGGTGAGCATCTTCGGCGTG195                                    GCCAGCCGGTTCTTCGTGGCCATGAGCAGC AAGGGCAAG234                                   CTCTATGGCTCGCCCTTCTTCACCGATGAGTGCACGTTC273                                    AAGGAGATTCTCCTTCCCAACAACTACAACGCCTACGAG312                                    TCCTACAAGTACCCCGGCATGTTCA TCGCCCTGAGCAAG351                                   AATGGGAAGACCAAGAAGGGGAACCGAGTGTCGCCCACC390                                    ATGAAGGTCACCCACTTCCTCCCCAGGCTGTGA423                                          (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 145                                                              (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE:                                                           (A) DESCRIPTION: Protein                                                      (ix) FEATURE:                                                                 (A) NAME/KEY:                                                                 (B) LOCATION:                                                                 (C) IDENTIFICATION METHOD:                                                    (D) OTHER INFORMATION: This sequence,                                          corresponding to bovine basic fibroblast                                     growth factor, can be found in Table 1,                                       page 9, lines 9, 14, and 19, in the                                           application, as filed.                                                        (x) PUBLICATION INFORMATION:                                                  (A) AUTHORS:                                                                  (B) TITLE:                                                                    (C) JOURNAL:                                                                  (D) VOLUME:                                                                   (E) ISSUE:                                                                    (F) PAGES:                                                                    (G) DATE:                                                                     (H) DOCUMENT NUMBER:                                                          (I) FILING DATE:                                                              (J) PUBLICATION DATE:                                                         (K) RELEVANT RESIDUES IN SEQ ID NO: 1-145                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       ProAlaLeuProGluAspGlyGlySerGlyAlaPheProProGlyHis                              15 1015                                                                       PheLysAspProLysArgLeuTyrCysLysAsnGlyGlyPhePheLeu                              202530                                                                        ArgIleHisProAspGlyArgVal AspGlyValArgGluLysSerAsp                             354045                                                                        ProHisIleLysLeuGlnLeuGlnAlaGluGluArgGlyValValSer                              5055 60                                                                       IleLysGlyValCysAlaAsnArgTyrLeuAlaMetLysGluAspGly                              65707580                                                                      ArgLeuLeuAlaSerLysCysValThrAspGluCysPhePhePhe Glu                             859095                                                                        ArgLeuGluSerAsnAsnTyrAsnThrTyrArgSerArgLysTyrSer                              100105110                                                                     SerTr pTyrValAlaLeuLysArgThrGlyGlnTyrLysLeuGlyPro                             115120125                                                                     LysThrProGlyGlnLysAlaIleLeuPheLeuProMetSerAlaLys                              130 135140                                                                    Ser                                                                           145                                                                           (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 140                                                               (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE:                                                           (A) DESCRIPTION: Protein                                                      (iii) HYPOTHETICAL: No                                                        (iv) ANTI-SENSE: No                                                           (ix) FEATURE:                                                                 (A) NAME/KEY:                                                                 (B) LOCATION:                                                                 (C) IDENTIFICATION METHOD:                                                    (D) OTHER INFORMATION: This sequence,                                         corresponding to bovine acid fibroblast                                       growth factor, can be found in Table 1,                                       page 9, lines 26, 31, and 36 in the                                           specification, as filed.                                                      (x) PUBLICATION INFORMATION:                                                   (A) AUTHORS:                                                                 (B) TITLE:                                                                    (C) JOURNAL:                                                                  (D) VOLUME:                                                                   (E) ISSUE:                                                                    (F) PAGES:                                                                    (G) DATE:                                                                     (H) DOCUMENT NUMBER:                                                          (I) FILING DATE:                                                              (J) PUBLICATION DATE:                                                         (K) RELEVANT RESIDUES IN SEQ ID NO: 1-140                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                      PheAsnLeuProLeuGlyAsnTyrLysLysProLysLeuLeuTyrCys                              151015                                                                        SerAsnGlyGlyTyrPheLeuArgIleLeuProAspGlyThrV alAsp                             202530                                                                        GlyThrLysAspArgSerAspGlnHisIleGlnLeuGlnLeuCysAla                              354045                                                                        GluSerIle GlyGluValTyrIleLysSerThrGluThrGlyGlnPhe                             505560                                                                        LeuAlaMetAspThrAspGlyLeuLeuTyrGlySerGlnThrProAsn                              6570 7580                                                                     GluGluCysLeuPheLeuGluArgLeuGluGluAsnHisTyrAsnThr                              859095                                                                        TyrIleSerLysLysHisAlaGlu LysHisTrpPheValGlyLeuLys                             100105110                                                                     LysAsnGlyArgSerLysLeuGlyProArgThrHisPheGlyGlnLys                              115120 125                                                                    AlaIleLeuPheLeuProLeuProValSerSerAsp                                          130135140                                                                     (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 423                                                               (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE:                                                           (A) DESCRIPTION: Genomic DNA                                                  (ix) FEATURE:                                                                 (A) NAME/KEY:                                                                 (B) LOCATION:                                                                 (C) IDENTIFICATION METHOD:                                                    (D) OTHER INFORMATION: This sequence                                          corresponds to K-FGF-140 and can be                                           found on page 11, lines 39-47 and page                                        12, lines 1- 31, in the application, as                                       filed.                                                                        (x) PUBLICATION INFORMATION:                                                  (A) AUTHORS:                                                                  (B) TITLE:                                                                    (C) JOURNAL:                                                                  (D) VOLUME:                                                                   (E) ISSUE:                                                                    (F) PAGES:                                                                    (G) DATE:                                                                     (H) DOCUMENT NUMBER:                                                          (I) FILING DATE:                                                               (J) PUBLICATION DATE:                                                        (K) RELEVANT RESIDUES IN SEQ ID NO: 1-423                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       GCGGCCGTCCAGAGCGGCGCCGGCGACTACCTGCTGGGC39                                     AlaAlaValGlnSerGlyAlaGlyAspTyrLeuLeuGly                                       15 10                                                                         ATCAAGCGGCTGCGGCGGCTCTACTGCAACGTGGGCATC78                                     IleLysArgLeuArgArgLeuTyrCysAsnValGlyIle                                       152025                                                                        GGCT TCCACCTCCAGGCGCTCCCCGACGGCCGCATCGGC117                                   GlyPheHisLeuGlnAlaLeuProAspGlyArgIleGly                                       3035                                                                          GGCGCGCACGCGGACACCCGCGACAGCCTGCTG GAGCTC156                                   GlyAlaHisAlaAspThrArgAspSerLeuLeuGluLeu                                       404550                                                                        TCGCCCGTGGAGCGGGGCGTGGTGAGCATCTTCGGCGTG195                                    SerPro ValGluArgGlyValValSerIlePheGlyVal                                      556065                                                                        GCCAGCCGGTTCTTCGTGGCCATGAGCAGCAAGGGCAAG234                                    AlaSerArgPhePheValAlaMe tSerSerLysGlyLys                                      7075                                                                          CTCTATGGCTCGCCCTTCTTCACCGATGAGTGCACGTTC273                                    LeuTyrGlySerProPhePheThrAspGluCysThrPhe                                       80 8590                                                                       AAGGAGATTCTCCTTCCCAACAACTACAACGCCTACGAG312                                    LysGluIleLeuLeuProAsnAsnTyrAsnAlaTyrGlu                                       95100                                                                          TCCTACAAGTACCCCGGCATGTTCATCGCCCTGAGCAAG351                                   SerTyrLysTyrProGlyMetPheIleAlaLeuSerLys                                       105110115                                                                     AATGGGAAGACCAAGAAGGGGAA CCGAGTGTCGCCCACC390                                   AsnGlyLysThrLysLysGlyAsnArgValSerProThr                                       120125130                                                                     ATGAAGGTCACCCACTTCCTCCCCAGGCTGTGA 423                                         MetLysValThrHisPheLeuProArgLeu                                                135140                                                                        (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                           (ii) FEATURE:                                                                (A) OTHER INFORMATION: This sequence can be                                   found on page 15, line 9, in the                                              application as filed.                                                         (iii) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                      GCGGCCGTCCAGAGCGGCGCCGGCGAC27                                                 (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 30                                                               (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) FEATURE:                                                                 (A) OTHER INFORMATION: This sequence can be                                   found on page 15, line 10, in the                                             application as filed.                                                         (iii) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                      ATGGCAGCAGTTCAATCAGGAGCAG GCGAC30                                             (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) FEATURE:                                                                 (A) OTHER INFORMATION: This sequence can be                                   found on page 15, line 11, in the                                              application as filed.                                                        (iii) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                      MetAlaAlaValGlnSerGlyAlaGlyAsp                                                1510                                                                      

What is claimed is:
 1. An isolated DNA having the sequence (SEQ IDNO:2:): ##STR10##